The Preliminary Investigation Of Molecular Mechanism Of Maize Roots Responsed To Low Phosphorus Stress

The deficiency of available phosphorus in soils is a globle problem and becoming an important limeted facor for agricultural production and crop yiled increase. Cultivation of crop varieties with high efficiency for phosphorus absorbing and utilization is one of the important approaches to solve the aboving problem. In the main maize production areas of China and other countries, the lack of available phosphorus is becoming an indisputable fact and restricts the maize production badly. The cultivation and extension of maize varieties with high efficiency for phosphorus absorbing and utilization is the vital guarantee of concept achievement on'low imput, environmentally-friendly with steady maize production'. So, screening and identification of maize phosohorus high efficieny germplasm resource, validation of important traits and key genes related to low phosphorus stress resopnse, verification of the molecular regulation mechanism of tolerance to low phosphorus and facilitated by traditional breeding methods and molecular biology technologies are important approaches for cultivation of maize varieties with effective, stability and safety. Based on the sreening and identification of maize inbred lines, this paper analyzed the changes of maize roots under low phosphorus stress, and then microarray and miRNA cloning methods were used for indentication of the different expression genes and related miRNAs in elite line 178 roots responsed to low phosphorus stress. Enzyme activity test, semiquantitativeRT-PCR and Real-time PCR analysis were applicated for further validation of some candidate genes. In summary, we have obtained the following results:1. The reduplicative identification of 20 maize inbred lines screened before in filed tolerance to low phosphorus stress by sand pot and field approaches and obtained in rough agreement with the before results. We have obtained 3 high phosphorus efficiency inbred lines as followings,178, RP125 and 99S2052-2 and other 10 susceptive lines to low phosphorus stress.2. The root characters were analyzed using 3 tolerant and 3 susceptive lines which random selected from 10 susceptive lines under low phosphorus stress. The results indicated that the absolute values of root total length, total number and total superficial area of high phosphorus efficiency inbred lines exceeded that of susceptive lines in sand pot. And in hydroponics, both the absolute values and growth rates of root total length, total number and total superficial area of high phosphorus efficiency inbred lines exceeded that of susceptive lines. 3. Affymetrix maize genome expression microarray was used for screening the different genes in maize roots of 178 under low phosphorus stress for 3 days.487 different expression genes were obtained and 349 genes were induced and 138 genes were repressed in response to low phosphorus stress respectively. These different genes were conducted for function annotation, which indicated that the functions of these genes were involvement in substance and energy metabolism, substances absorption and transport, transcriptional regulation, stress response, phytohormone signal transduction, electron transfer and other processes related to growth and development.4. The expression patterns of maize inorganic phosphorus transporters, acid phosphatase, phytase,2-deoxymugineic acid synthasel, POD and MYB transcription factor were validated in 178 roots responsed to low phosphorus stress by Real-time PCR. The results indicated that the expression tendencies of these candidate genes were in rough coincident with the result of microarray analysis and with different expression patterns among these genes after the on set of low phosphorus stress.5. Phosphorus content and related enzyme activities including Apase(inner and outside), phytase, POD and the activity of root in maize roots were analyzed using inbred line 178 with the identical treated time like aboving material. The results indicated the content of phosphorus was decreased and the inner activity of Apase was induced, but the outside activity of Apase was changed unnoticeably under low phosphorus. Also, the activity of POD was increased and the activity of phytase was ascended placidly, and the activity of root was decreased under low phosphorus.6. We have successfully constructed a small RNA library of 178 roots from 6 time points under low phosphorus stress.263 clones were obtained by RT-PCR detection and selected for sequencing analysis. Sequence blast and miRNA structure prediction indicated that a total of 12 sequences were conforming to the evaluation criterion of miRNAs. Further analysis found that 2 miRNA was identical with the sequence of maize known miRNAs (miRNA156 and miRNA399), and other miRNAs were deemed to new miRNAs in maize.7. The conservation of these candidate miRNAs among plant species were analyzed by bioinformatic tools. The results indicted that exception of the two known miRNAs were conserved among plant species, another new cloned miRNA, Zm-miRNA2 was found to be conserved among gramineous species. Simultaneity, Zm-miRNA9 was also found to be with the identical sequence with known maize miRNA169 family members'exception of specific base and was falled under new member of miRNA169.8. The authenticities and expression patterns of these 12 miRNAs were analyzed by polyA-tailed RT-PCR. It indicated that exception of Zm-miRNA6, which was detected only in 12 h,24 h, and 48 h treated time point, other miRNAs could be detected at the majority of treated time point (0h,12 h,24 h,48 h,72 h, and 96 h). Expression pattern indicated that Zm-miRNA2, Zm-miRNA3, Zm-miRNA5, Zm-miRNA6, Zm-miRNA9, and miRNA399b could be induced and Zm-miRNA8 and Zm-miRNA10 could be repressed under low phosphorus stress. Other two miRNAs, Zm-miRNA8 and Zm-miRNA10 may be induced first then repressed. However, the expression of miRNA156 was no significant change.9. The targets of 12 miRNAs were predicted by web based miRNA prediction software. The results were indicated that a total of 57 putative target genes were predicted for 11 miRNAs, except of Zm-miRNA9. These genes were referring to diverse gene families and different biological functions including substances and energy metabolism, transcription regulation, transport, stress response, signal transduction and so on. Further analysis found that some of these genes were also found with different expression in microarray analysis. For example, maize inorganic transporters (TC420181) were targeted by miRNA399b and Zm-miRNA3. Zm-miRNA2, Zm-miRNA5 and Zm-miRNA6 target bZIP (EE023309), ubiquitin carrier protein (TC432098) and GST (TC385801), respectively.10. The expression patterns of miRNA399, Zm-miRNA3 and theirs targets, ZmPT1/2 were analyzed by Real-time PCR, which indicated that expression pattern of miRNA399b was induced obviously in response to low phosphorus stress. The expression pattern of Zm-miRNA3 could be induced first, and then went mild and the overall trendency was induced.11. Based on the known regulatory mechanism and signal pathway in arabidopsis and rice in response to low phosphorus, the putative adaptation mechanism of maize root was investigated and the regulatory models of metabolism pathway and signal transduction was simulated according to the result of microarray and miRNA target prediction. These findings could be used for reference for further understanding the whole molecular mechanism and regulation processes mediated by miRNAs in maize roots responsed to low phosphorus stress.